In vitro assays for inhibition of microglial activation

ABSTRACT

The present invention provides cell-based assays, including high throughput cell-based assays, for identification of candidate therapeutic agent with no known agents with the ability to inhibit microglial activation in vivo in response to different ligands.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NS052189awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the field of compositions and in vitroassays for the identification of agents for the prevention and treatmentof pathological conditions associated with inflammation and/or neuronalinjury.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an admission of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Microglia, the resident innate immune cells in the brain, have long beenimplicated in the pathology of neurodegenerative diseases.Neurodegenerative diseases (ex. Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, ALS, etc.) share common characteristics,such as changes in microglial number and morphology, elevated cytokinelevels, oxidative stress, and progressive neuronal loss. Increasingevidence reports that microglia can become a chronic source of cytokinesand reactive oxygen (ROS) that drive progressive neuron damage and areimplicated in the chronic nature of neurodegenerative diseases. Block ML and Hong J S. Biochem Soc Trans. 2007; 35:1127-1132.

Microglia can become chronically activated by either a single stimulus,e.g., Lipopolysaccharide (“LPS”) stimulus or neuron damage, or bymultiple stimuli exposures to result in cumulative neuronal loss overtime. Activated microglia produce toxic factors (cytokines and reactiveoxygen species) following either single or chronic exposure to diseaseproteins, environmental toxins, cytokines, and neuronal damage,resulting in the progressive loss of neurons over time, a fundamentalcomponent of neurodegenerative disease. Lull M E and Block M L,Neurotherapeutics. 2010 October; 7(4): 354-365. Recent work suggeststhat redox signaling in microglia may be a critical mechanism of chronicneuroinflammation, and certain cells may be inherently vulnerable toreactive microgliosis id. While the mechanisms driving these phenomenaare just beginning to be understood, the microglial response to neurondamage (“reactive microgliosis”) and ROS have been implicated as keymechanisms of chronic and neurotoxic microglial activation.

There is thus a need in the art for a method of identifying candidatetherapeutic agents with the ability to inhibit such microglialactivation in pathologies associated with neuronal injury. The presentinvention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides cell-based assays, includinghigh-throughput cell-based assays, for identification of candidatetherapeutic agents with the ability to inhibit microglial activation invivo.

In one embodiment, the invention provides a cell-based assay foridentification of candidate therapeutic agents that inhibit microgliaactivation in vivo, comprising providing in vitro activated microglialcells, contacting said activated microglial cells with one or more testagents, identifying one or more test agents that exhibit inhibition ofthe in vitro microglia activation, and determining toxicity of theidentified test agents that exhibit inhibition of the in vitro microgliaactivation by determination of cell death upon exposure of the testagents to the microglial cells. The test agents that exhibit inhibitionof the in vitro microglia activation, and lack of toxicity, arecandidate therapeutic agents for the inhibition of microglial celldeath.

The extent of microglial activation in the invention can be measured ona percentage basis, and thus agents with a particular percentage levelof inhibition of microglial activation can be identified. In theparticular examples of the invention, agents are identified with atleast a 50% level of inhibition of microglial activation using thecell-based assay of the invention. The stringency of such inhibition canbe increased or decreased depending upon the specific criteria andparameters applied to an experiment using the cell-based assays. Thus,although the identified candidate therapeutic agents described hereinwere identified based on the criteria of 50% or greater inhibition ofmicroglial activation in the cell-based assays of the invention, aperson of skill in the art could also identify candidate therapeuticagents that have either higher or lower levels of inhibition ofmicroglial activation, as demonstrated in the examples and accompanyingfigures herein.

The invention also provides high-throughput cell-based assays foridentifying a candidate therapeutically active agent for clinicalinhibition of microglia activation, comprising the steps of providing apanel of in vitro activated microglial cells, contacting said panel ofmicroglial cells with one or more individual test agents, determiningwhether the test agents cause inhibition of the activation of themicroglial cells treated with the test agents, and determining the levelof cell death of the microglial cells treated with the test agents,where the one or more test agents that exhibit inhibition of theactivation of the microglial cells and no increased cell death arecandidate therapeutically active agents.

The microglial cells used in the assays of the invention may be primarymicroglia or they may be cells from an immortalized microglia cell linethat display the requisite physiological attributes for use in thecell-based assays. The microglia used in the assays of the invention maybe activated using a variety of in vitro mechanisms. This includesactivation of microglia by in vitro exposure to activators of microglia,e.g., soluble ligands or immobilized substrates, including bindingpartners of CD11b/CD18 and TLR4. For example, in some aspects, themicroglial cells are activated by in vitro exposure to an active fibrincomposition. In another example, the microglial cells are activated byin vitro exposure to LPS, a gram negative bacterial immunostimulant thattriggers a cascade of proinflammatory events that mimic pathologicalresponses. It will be apparent to one skilled in the art upon readingthe present invention that various mechanisms of activating microglialcell in vitro can be used with the cell-based assays of the presentinvention.

The activation of the microglia in the cell-based assay of the inventionmay be determined using a number of biochemical and morphological endpoints. For example, microglial activation may be measured using geneexpression, e.g., increased expression of TNF, IL-1, Clcx10, Ccl5, IL-10and/or Mcp-1. In another example, microglial activation can beidentified through size of the microglia, with activated microgliagenerally having a surface area of ≧800 μm².

Preferably, the assays of the invention also measure the toxicity oftest agents to identify candidate therapeutic agents that inhibitmicroglia activation but which do not cause microglia cell death.Toxicity through cell death can be identified in the assay, e.g., byidentification of the hallmark apoptotic DNA ladders or by morphologicalchanges, such as shrinkage of the cell surface area of the microglia. Inone specific aspect, cell death is identified in the cell-based assaysthrough shrinkage of the cell surface area of the microglia to 150 μm²or less.

Various test agents can be used in the cell-based assays of the presentinvention. In some embodiments, the test agents used in the screeningare agents with demonstrated affinity to targets involved in microgliaactivation. In other embodiments, the test agents used in the screeningare agents with no known activity or identified binding target. In stillother embodiments, the test agents used in the screening are agents thatcomprise known drugs with clinical evaluation data. In still otherembodiments, the test agents used in the screening are agents thatcomprise known anti-inflammatory and microglia-macrophage responsemodulators.

One advantageous feature of the assays of the present invention is theability of the assays to identify ligand-selective inhibitors ofmicroglial activation in a high throughput fashion.

Another feature of the invention is that the cell-based assays allowdirect comparisons of compound selectivity of the various agents testedfor inhibition of microglia activation.

Another advantage of the assays of the invention is that they can beused to screen different classes and varieties of agents, includingantibody, peptide or small molecule libraries.

These assays can be used in primary screens to discover novel inhibitorsof innate immunity, as well as in secondary screens to assess ligandspecificity or to provide additional data on the predicted functionalactivity of the test agents in an in vivo setting for therapeutic use.

Yet another feature of the invention is the ability to utilizemorphologic activation characteristics as the outcome measure of theassays. All prior methods of identification of ligand-selectiveinhibitors were either based on adhesion of cell lines or non-cellularassays with little predictive value for in vivo efficacy. Suchinhibition of morphologic activation in vitro has been shown to havepredictive value for in vivo efficacy (Adams R A et al., J Exp Med, 2007Mar. 19; 204(3):571-82).

An advantage of the cell-based assays of the invention are that they arewell suited for a high throughput screening (HTS) system, and theinvention has demonstrated that the assay can be fully automated using96 or 384-well plates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating the basic steps involved in usingcell-based microglial activation assays to identify candidatetherapeutic agents.

FIG. 2 is a graph showing the assay performance for a first screen ofagents for inhibition of fibrin-induced microglia activation.

FIG. 3 is the summary of the amount of cell death within the normalrange for test agents and negative and positive controls in the firstscreen of agents for inhibition of fibrin-induced microglia activation.

FIG. 4 is a plot showing the distribution of percent inhibition valuesfor tested agents and positive and negative controls in the HTS ofagents that inhibit in vitro fibrin-activation of microglia.

FIG. 5 is a set of plots showing the range of activity of the testedagents in the fibrin inhibition assay, including the 128 determined tobe “hits” based on inhibited microglia activation and lack of toxicityin the microglia fibrin-activation inhibition HTS.

FIG. 6 is a graph showing the assay performance for a first screen ofagents for inhibition of LPS-induced microglia activation. Z′ values forthe inhibitors of LPS-induced percent microglia activation were between0.47 and 0.56 for the six assay plates.

FIG. 7 is the summary of the amount of cell death within the normalrange for test agents and negative and positive controls in the HTS ofagents that inhibit in vitro LPS-activation of microglia.

FIG. 8 is a set of plots showing the range of activity of the testedagents, including the 146 determined to be “hits” based on inhibitedmicroglia activation and lack of toxicity in the microgliaLPS-activation inhibition HTS.

FIG. 9 shows the distribution of hit compounds efficacious against bothFibrin and LPS-induced microglia activation.

DETAILED DESCRIPTION OF THE INVENTION

The methods described herein may employ, unless otherwise indicated,conventional techniques and descriptions of molecular biology (includingrecombinant techniques), cell biology, biochemistry, and microarray andsequencing technology, which are within the skill of those who practicein the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of oligonucleotides, sequencing ofoligonucleotides, and detection of hybridization using a label. Specificillustrations of suitable techniques can be had by reference to theexamples herein. However, equivalent conventional procedures can, ofcourse, also be used. Such conventional techniques and descriptions canbe found in standard laboratory manuals such as Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, 1988; Sambrook and Russell, Molecular Cloning: A Laboratory Manual(2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L.,Biochemistry (4th Ed.) W.H. Freeman, New York (1995); Lehninger,Principles of Biochemistry, 3^(rd) Ed., W. H. Freeman Pub., New York(2000); and Berg et al., Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York (2002), all of which are herein incorporated by reference intheir entirety for all purposes. Before the present compositions,research tools and methods are described, it is to be understood thatthis invention is not limited to the specific methods, compositions,targets and uses described, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present invention, which will be limited only by appendedclaims.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atest agent” refers to one, more than one, or mixtures of such agents,and reference to “a method” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that eachintervening value between the upper and lower limit of that range—andany other stated or intervening value in that stated range—isencompassed within the invention. Where the stated range includes upperand lower limits, ranges excluding either of those included limits arealso included in the invention.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing the formulations and methodologiesthat are described in the publication and which might be used inconnection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

DEFINITIONS

The term “antibody” is intended to include any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. As antibodies can be modified in a number of ways, the term“antibody” should be construed as covering any specific binding memberor substance having a binding domain with the required specificity.Thus, this term covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially synthetic. Where bispecific antibodies are to be used,these may be conventional bispecific antibodies, which can bemanufactured in a variety of ways (Holliger and Winter, Curr OpinBiotechnol. 1993 August; 4(4):446-9), e.g., prepared chemically or fromhybrid hybridomas, or may be any of the bispecific antibody fragmentsmentioned above. It may be preferable to use scFv dimers or diabodiesrather than whole antibodies. Diabodies and scFv dimers can beconstructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction. Other formsof bispecific antibodies include the single chain “Janusins” describedin Traunecker A et al., EMBO J. 1991 December; 10(12):3655-9. Suchantibodies also include CRAbs, which are chelating antibodies whichprovide high affinity binding to an antigen, D. Neri, et al. J. Mol.Biol, 246, 367-373, and dual-variable domain antibodies as described inWu C et al., Nat Biotechnol. 2007 November; 25(11):1290-7. Epub 2007Oct. 14.

The term “microglial activation” as used herein can refer to processesassociated with innate activation or adaptive activation of themicroglia. Such activation may include morphological changes of themicroglial cells, including shortening of cellular processes andenlargement of their soma, as well as the release of proinflammatorycytokines and chemokines, reactive oxygen and/or nitrogen intermediates,proteinases and complement proteins, and upregulation of cell surfaceactivation antigens.

The term “neurodegeneration” refers to a physiological state caused byneuronal injury associated with neuronal loss and/or damage. In specificaspects, neurodegeneration refers to neuronal injury resulting inimpaired cognitive function.

The term “neuronal injury” as used herein refers to any damage ordysfunction exhibited by neurons, including but not limited to loss ofmyelin, dendrite retraction, dendritic spine density reduction, axonaldamage and neuronal death. A “neuronal condition” as used herein refersto any damage or dysfunction exhibited by neurons, including but notlimited to dendrite retraction, dendritic spine density reduction,axonal damage and neuronal death.

The term “pharmaceutically acceptable carrier” as used herein isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and agents is well known in the art.Except insofar as any conventional media or agent is incompatible withthe agents provided herein, use thereof in the composition iscontemplated.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified, labeled or derivatized aminoacids, and polypeptides having modified peptide backbones.

The term “peptidomimetic” as used herein refers to a protein-like chaindesigned to mimic a peptide. They typically arise from modification ofan existing peptide in order to alter the molecule's properties. Forexample, they may arise from modifications to change a molecule'sstability, biological activity, or bioavailability.

The term “pharmacophore” is used herein in an unconventional manner.Although the term conventionally means a geometric and/or chemicaldescription of a class or collection of compounds, as used here the termmeans a compound that has a specific biochemical activity or bindingproperty conferred by the 3-dimensional physical shape of the compoundand the electrochemical properties of the atoms making up the compound.Thus, as used here the term “pharmacophore” is a compound and not adescription of a collection of compounds which have definedcharacteristics. Specifically, a “pharmacophore” is a compound withthose characteristics.

The term “small molecule” refers to a molecule of a size comparable tothose organic molecules generally used in pharmaceuticals. The termexcludes biological macromolecules (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000 Da, and most preferably up to about 1000Da.

A “test agent” as used herein refers to any agent that is a candidate totreat a disease or symptom thereof. Such agents include, but are notlimited to, peptides; proteins (including derivatized or labeledproteins); antibodies or fragments thereof small molecules; aptamers;carbohydrates and/or other non-protein binding moieties; derivatives andfragments of naturally-occurring binding partners; peptidomimetics; andpharmacophores.

As used herein, the terms “treat,” “treatment,” “treating,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment,” as used herein,covers any treatment of a disease in an animal, particularly in a human,and includes: (a) preventing the disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, e.g., causing regression ofthe disease, e.g., to completely or partially remove symptoms of thedisease.

The Invention in General

The present invention is based on the novel development of a compositionthat mimics the biochemical and proinflammatory properties of fibrinmatrices found in vivo during pathological conditions in an in vitrosetting. These fibrin compositions are highly reproducible in activity,and provide the advantage of essentially mimicking the in vivo activityof fibrin, and use of such compositions in assays enable the screeningof fibrin inhibitors that have a higher likelihood of being efficaciousin clinical use. More particularly, by using a fibrin composition withsimilar properties of fibrin in vivo activity observed in pathologicalconditions, the probability of finding biologically active inhibitorswith in vivo efficacy is enhanced.

Microglia are activated in response to injury and immunological stimuli,and in doing so undergo dramatic alterations in morphology. See, e.g.,Kreutzberg G W. Trends Neurosci. 1996; 19:312-318. It has been shown,however, that changes in morphology alone are unlikely to allow adifferentiation between microglial activation in response to normal,physiological changes and toxic microglial activation associated withpathological states such as neurodegeneration. Lynch M A. Mol Neurobiol.2009; 40:139-156. In reacting to extracellular signals, such as thepresence of pathogens, foreign material and dead or dying cells,microglia may undergo a morphological change into an amoeboid shape withshort or non-existent processes. This morphological change is alsoaccompanied by changes in signaling and gene expression that can resultin changes in surface receptor expression, the release of pro- oranti-inflammatory factors, recruitment molecules, and ROS, among others.The cumulative effect of these changes in morphology and phenotype is ashift from resting to activated microglia.

Although microglia and macrophages are different cell types, they shareseveral common pro-inflammatory pathways. Therefore the cell-basedassays of the invention can also be used in the discovery of inhibitorsfor macrophage activation.

The central aim of the present invention was to provide a novelhigh-content, high throughput assay that could measure the activation ofprimary microglia in response to activators such as LPS, or otheractivators (e.g. fibrin, complement, amyloid) in a format that wouldallow efficient and large-scale screening, e.g., of small moleculeand/or antibody libraries.

In designing the high throughput screening assays of the invention,fibrin and LPS were selected as two ligands that activate microgliathrough different receptors, CD11b/CD18 and TLR4, respectively.Microglia cells will change morphology upon activation by LPS, includingretracting cell processes and becoming amoeboid, with a several foldincrease in cell surface area. This morphological change upon activationallows the development of a fully automated high content HTS that usesmicroglial cell surface area measurement as a quantification ofactivation of microglia.

A significant increase in cell size is one of the most prominentfeatures of microglia activation and correlates with increasedpro-inflammatory activity. The inventors developed a fluorescent assayfor quantification of changes in microglial cell size and used theINCell Analyzer 2000™ instrument (GE Healthcare, San Francisco, Calif.)for image acquisition and analysis. The assay was adapted to a 96-wellformat to evaluate assay conditions and automated image acquisitionanalysis with the INCell instrument and software and then miniaturizedto a 384-well plate format, using fully automated coating of themicroglial activating agent (e.g., fibrin), plating of microglia cells,addition of compounds, addition of activator (e.g., LPS), as well as thecomponents for the fixation and staining procedures necessary forconsistent measurement of cell morphology. The protocol for theisolation of rat primary microglia yield enough cells for seven to eight384 well assay plates per litter of rat pups.

Primary Screening of Test Agents

The cell-based assays of the invention can be used to identify testagents such as small-molecule or monoclonal antibody inhibitors ofmicroglial cell activation that act through various targets andmechanisms. By measuring phenotypic cell end points rather than e.g.,binding to a specific target or modulation of expression of a particulargene, identification of test agents that may modulate complex pathwaysand/or target the effects of multiple pathways or targets simultaneouslycan be performed. Thus, in cases where the underlying biologicalmechanisms are not yet fully understood, such as neurodegenerativediseases, the ability to identify the inhibition of biological processessuch as microglial activation provides a significant advantage overpurely biochemical assays.

In certain embodiments, the library of test agents used for screening inthe cell-based assays of the invention is from a focused library ofagents with known anti-inflammatory properties or known to modulatemicroglial/macrophage response. Such molecules may not be confirmed astarget-based agents, but may provide an enriched population of agentswith the potential of having a physiological effect on microgliaactivation.

Secondary Screening in Target-Based Drug Discovery

The cell-based assays of the invention can also play a critical role intarget-based drug discovery as confirmatory functional assays forcompounds identified by primary biochemical screening. If a target isknown to be involved in the activation of microglia, a phenotypic assaysuch as those described herein would allow testing the effects of theagents that have demonstrated affinity to such targets in aphysiologically relevant context. Following identification of aninhibitor of a target identified as involved or potentially involved inthe activation of microglia, a cell-based assay of the present inventionallows confirmatory testing of these test agents in a biologicalcontext.

Microglial Cells for Use in the Assays of the Present Invention

The cells for use in the assays of the invention include primarymicroglia cells and immortalized cell lines. Rodent cell lines that areuseful in the assays of the invention include, but are not limited to,BV-2, C8-B4, N9, N11, various EOC lines, highly aggressive proliferatingimmortalized (HAPI) cell lines, and MG5. Horvath, R J et al., J.Neurochem. 2008, 107, 557-569; Ohsawa, K. et al. Glia 1997, 21, 285-298;Stansley, B et al. J. Neuroinflammation 2012, 9, 115. Human immortalizedmicroglial cell lines, such as HMO6 may be preferred for certainneurotherapeutic studies, as their phenotype is more closely related toprimary human microglia. Nagai, A et al., J. Neurosci. Res. 2005, 81,342-348. Experimental evidence shows that compared with cell lines,primary microglial cells more closely resemble both the phenotype andthe stimulus responses of microglial cells in vivo. Stansley et al., Id.One exemplary method for obtaining a population of primary microglialcells consists of establishing a confluent mixed glial culture from thebrains of neonate rodents. Isolation of the microglia can beaccomplished by culturing the primary cells and collecting the detachedcells. Giulian, D and Baker, T J J. Neurosci. 1986, 6, 2163-2178. Morerecently, it has been possible to produce microglial cells from stemcells using a modified neuronal differentiation method. Beutner, C etal. Nat Protoc. 2010, 5, 1481-1494. Microglia may also be isolated fromhuman iPS cells. From a pathophysiological point of view, thesemicroglial cells are more relevant than immortalized cell lines due totheir similarity to freshly isolated primary microglia, essentiallydisplaying indistinguishable morphology, functions, and cellularmarkers. Since this cell source potentially offers an unlimited supplyof microglia generated in vitro, this may be a particularly attractivecell source for the high throughput screening assays of the presentinvention.

Microglia Activation in Neurodegenerative Disorders

As the brain's resident macrophage, microglial cells play a key role inprotection against exogenous and endogenous insults. Disruption of brainhomeostasis caused by physiological or pathological conditions inducesmicroglial cell activation and the release of cytokines, chemokines, andtoxins that lead to inflammation. This appears to be a common process ina number of acute and chronic neurodegenerative diseases such as stroke(Morioka, T et al., J. Comp. Neurol. 1993, 327, 123-132), Alzheimer'sdisease (Gao, H M and Hong, J S, Trends Immunol. 2008, 29, 357-365),Parkinson's disease (McGeer, P L et al., Neurology 1988, 38, 1285-1291),Multiple sclerosis (McGeer, P L et al., Neurology 1988, 38, 1285-1291),ALS (Banati, et al., Clin. Neuropathol. 1995, 14, 197-200), Huntingtondisease (Moller, T. J. Neural Transm. 2010, 117, 1001-1008), and HAND(Zindler, E and Zipp, F Best Pract. Res. Clin. Anaesthesiol. 2010, 24,551-562). Microglial activation can induce or exacerbate such neuronaldamage, and postmortem analyses of human tissues and animal models ofslow progressing neurodegenerative diseases have shown neuronal loss inconjunction with increased levels of activated microglia in areas of thebrain with lesions. Polazzi, E and Monti, B. Prog. Neurobiol. 2010, 92,293-315; Boillee, S et al., Science 2006, 312, 1389-1392. The cell-basedassays of the invention thus may be particularly useful to identifycandidate therapeutic agents for the treatment of such disorders.

Test Agents for Screening Using the Assays of the Invention

The assays of the invention are used to identify candidate therapeuticagents for the modulation of microglial activation in vivo. Thisincludes the testing of new agents as well as assays to test knowncompounds (including synthetic, recombinant or naturally-occurringcompounds) for their effect on microglial activation and/or cell death.

It is known in the pharmaceutical arts that binding affinity to a targetand efficacy do not necessarily correlate, and that identification ofcell-based activity changes conferred by a test agent is an improvedfunctional predictor of therapeutic activity compared to agentsidentified merely by affinity, e.g., binding of agents to microglialreceptors.

In certain aspects, the assays of the invention correlate with in vivomodulation of signaling through activated fibrin. Examples of cell-basedassays for use with the present invention include, but are not limitedto, high throughput binding screening; assays to measure cellactivation, proliferation, necrosis and/or apoptosis; flow cytometryassays; metabolic assays measuring labeling or turnover; phase andfluorescence microscopy; receptor phosphorylation and/or turnover; cellsignaling assays; immunohistochemistry studies; reporter gene assays,and subcellular fractionation and localization. More specific examplesof such assays are: FLIPR to detect changes in intracellular calciumconcentration and cell-based ELISA assays to detect and quantifycellular proteins including post-translational modifications associatedwith cell activation.

Biochemical assays can also be used to correlate binding with efficacyin the cell-based assay methods of the invention. These include, but arenot limited to, spectrophotometric assays, fluorometric assays,calorimetric assays, chemiluminescent assays, radiometric assays,chromatographic assays, colorimetric assays, and substrate specificityinhibitor kinase assays. Specific examples are: luciferase assays, inwhich firefly luciferase protein catalyzes luciferin oxidation and lightis generated in the reaction, and which is frequently used as a reportergene for measuring promoter activity or transfection efficiency;electrophoresis; gas-liquid chromatography; and Forster resonance energytransfer (FRET).

To confirm the functional activity of a test agent, a therapeuticallyeffective amount of a test agent of the invention may be administered toa subject (including an animal model of a neurological pathology) toconfirm its in vivo activity following identification in an assay of theinvention. By “therapeutically effective dose or amount” or “effectiveamount” is meant an amount of the test agent that, when administered,brings about a positive therapeutic response with respect to neuronalinjury. In some embodiments of the invention, the therapeuticallyeffective dose is in the range from about 0.1 μg/kg to about 100 mg/kgbody weight, about 0.001 mg/kg to about 50 mg/kg, about 0.01 mg/kg toabout 30 mg/kg, about 0.1 mg/kg to about 25 mg/kg, about 1 mg/kg toabout 20 mg/kg, about 3 mg/kg to about 15 mg/kg, about 5 mg/kg to about12 mg/kg, about 7 mg/kg to about 10 mg/kg or any range of value therein.It is recognized that the method of treatment may comprise a singleadministration of a therapeutically effective dose or multipleadministrations of a therapeutically effective dose.

The test agent is administered to supply a desired therapeutic dose topromote a desired therapeutic response of the modulator to thetherapeutic area. By “desired therapeutic response” is intended animprovement in the condition or in the symptoms associated with thecondition, including the inhibition of angiogenesis.

The test agents can be formulated in a unit dosage such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable carrier. Such carriers are inherently nontoxic andnontherapeutic. Examples of such carriers are saline, Ringer's solution,dextrose solution, and Hanks' solution. Nonaqueous carriers such asfixed oils and ethyl oleate may also be used. The vehicle may containminor amounts of additives such as substances that enhance chemicalstability, including buffers and preservatives.

Various methods of delivery can be used to deliver the test agent, andwill in part be dependent upon the agent and its bioavailability. Forexample, small molecules or other agents that are bioavailable may beadministered orally, whereas protein-based agents are generally but notexclusively administered parenterally. Certain agents may beadministered systemically, while others may be more beneficial with alocal delivery. The method of delivery will be apparent to one skilledin the art upon reading the specification, and can be determined in viewof the specific properties of the test agent.

It is understood that the effective amount of a test agent may varydepending on the nature of the effect desired, frequency of treatment,any concurrent treatment, the health, weight of the recipient, and thelike. See, e.g., Berkow et al., eds., Merck Manual, 16th edition, Merckand Co., Rahway, N. J. (1992); Goodman et al., eds., Goodman andGilman's The Pharmacological Basis of Therapeutics, 8th edition,Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3rdedition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987),Ebadi, Pharmacology, Little, Brown and Co., Boston (1985), Katzung,Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn.(1992), which references and references cited therein, are entirelyincorporated herein by reference.

The test agent may be contained in a pharmaceutically-acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and agents for delivering cells is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the cells or polypeptides provided herein, use thereofin the compositions is contemplated. Supplementary active compounds canalso be incorporated into the test agents.

For confirming in vivo activity, a composition comprising a test agentis formulated to be compatible with its intended route ofadministration. Examples of routes of administration includeintravenous, intraarterial, intracoronary, parenteral, subcutaneous,subdermal, subcutaneous, intraperitoneal, intraventricular infusion,infusion catheter, balloon catheter, bolus injection, direct applicationto tissue surfaces during surgery, or other convenient routes.

Solutions or suspensions used for such administration can include othercomponents such as sterile diluents like water for dilution, salinesolutions, polyethylene glycols, glycerin, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Thecomposition can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. Test agents suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, or phosphate buffered saline (PBS). In all cases,the composition must be sterile and should be fluid to the extentpossible. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents.

Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin. Sterile injectable solutionscan be prepared by incorporating an active agent in the required amountin an appropriate solvent with a selected combination of ingredients,followed by filter sterilization. Generally, dispersions are prepared byincorporating an active agent into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In many cases, it will be preferable to includeisotonic agents.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1 Isolation and Culture of Microglia Cells

Rat cortices were dissected from 2-3 postnatal day (P) old rat pups andtransferred into Dulbecco's Phosphate Buffered Saline (DPBS), containingglucose and sodium-pyruvate (Life Technologies/GIBCO, Grand Island,N.Y.). The meninges were removed and the cortices transferred into 7.5ml of the same DPBS (10 cortices) and minced with a sterile razor blade.Tissue pieces with DPBS were transferred into a 15 ml tube, 1.5 ml of2.5% Trypsin solution (10× Trypsin, Life Technologies/GIBCO, GrandIsland, N.Y.) and 500 μl of DNAse (SIGMA, D4513, 2000 Kunitz units/mewere added.

Following incubation at 37° C. for 25 minutes, the trypsin solution wasremoved, cortices were washed with 30% Fetal Bovine Serum (FBS) in DPBSand serially triturated with a 5 ml pipette with 7 ml of 30% FBS in DPBScontaining DNAse (1000 Kunitz units). The cell suspension was gentlyspun at 200×g for 15 minutes and the pellet re-suspended in DMEM (LifeTechnologies/GIBCO, Grand Island, N.Y.), containing 10% heat-inactivatedFBS (Life Technologies/GIBCO, Grand Island, N.Y.; lots pre-tested formicroglia survival), 100 units/ml Penicillin and 100 mg/ml Streptomycin(100× solution, Life Technologies/GIBCO, Grand Island, N.Y.). Cells areplated into Poly-D-lysine pre-coated T-75 flasks in 15 ml of medium, 2-3cortices per flask. Cells were incubated at 37° C. in a humidified 5%CO2 incubator. On day 3 in vitro, 5 ml of fresh medium were added andcells were grown for one more day.

On day 4 in vitro, flasks were placed onto a shaker platform, pre-heatedto 37° C. and microglia cells were shaken off the cortical cell layer at200 rpm for 2 hours. The medium containing mostly microglia cells wasremoved from the flasks and cells are spun at 200×g for 15 minutes. Thecell pellets were gently re-suspended in 200 μl of culture medium perpellet first to prevent cell clumping, then combined into one tube andfurther diluted to 10 ml. After counting the number of cells with ahematocytometer, the cell concentration was adjusted to approximately5000 cells/μL. The yield per rat cortex should amount to approximately2.5×10⁶ microglia cells.

Example 2 Production of Fibrin Assay Plates

A 6 μg/ml fibrin solution was used to coat the wells of a 384-wellplate. Microglia cells were further diluted with culture medium to6,000/50 μI per well and plated either onto the fibrin-coated wells of atest 384-well plate, or onto Poly-D-Lysine pre-coated black-walled384-well plates (μclear, LPS assay, Greiner, BioOne, Germany), using theThermo Scientific Matrix WellMate™ instrument at a medium speed setting(Thermo Scientific, Waltham, Mass.).

One hour after plating, 100 nl of compounds were added to each well from10 mM stock solutions in DMSO to a 10 μM final concentration, using aBiomek® automated laboratory workstation (Beckman-Coulter, Brea,Calif.). 50 μl of a 1 ng/ml solution of LPS, a known microgliaactivator, was added 1 hour after the addition of compounds, to a finalconcentration of 500 pg/ml, using the BioTek El-406™ liquid handlingsystem (Thermo Fisher, Waltham, Mass.). The liquid was added against thewall of each well to prevent dislodging of cells from the well surface.Cells were transferred to a 37° C., 5% CO₂ incubator for 48 hours.

50 μl of medium were removed from each well and 50 μl of 8%Paraformaldehyde were added using the BioTek El-406™ liquid handlingsystem. After incubation for 1 hour all liquid (100 μl) was removed andwells were washed 3 times with 100 μl of DPBS with calcium and Magnesium(Life Technologies/GIBCO, Grand Island, N.Y.). 50 μl of a 0.1%Triton-x-100 solution in DPBS was added and incubated for 1 hour. Afterincubation, wells are washed 3 times with DPBS. 50 μl of DPBS containing0.5 μg/ml CellMask™ Red Stain and 2 μg/ml Hoechst nuclear dye (LifeTechnologies/Invitrogen, Grand Island, N.Y.) are added and incubated for1 hour.

Example 3 Assay Segmentation of Activated and Dead Microglia Cells

Images of microglia cells were acquired with the INCell Analyzer 2000(GE Healtchare), using a 10× lens and excitation/emission filter pairsof 350 nm/455 nm (CellMask™ Red Stain) and 579 nm/624 nm (Hoechst dye).Images were analyzed with the GEHC INCell Developer Toolbox version 1.9.The Hoechst dye-stained cell nuclei were segmented using a “nuclear”segmentation type, with a set minimum target area of 30 μm² and setsensitivity of 75%. To minimize artifacts, any segmentation with lessthan 120 intensity units or area greater than 1000 μm² were excluded.The Cell Mask™ Red-stained whole microglia cells were segmented using anintensity segmentation type, with a set threshold between 200-4095intensity units. The borders of adjacent contacting cells were resolvedusing the “clump breaking” segmentation post-processing that utilizesdiscrete nuclei as seeds. Only cells containing a nucleus within thecell body area were used further in the screening assay. All segmentednuclei and cells were then recorded as individual counts. Activatedmicroglia cells were segmented by size into cells ≧800 μm². Dead cellswere segmented into cells <150 μm².

When microglia are treated with doses of LPS of 1 ng or higher resultingin cell death, all cells that were labeled with CellMask™ Red Stainretained the label, but shrink to a cell surface area of 150 μm² orless. This is an important measurement that can be used to assesscompound toxicity during screening for inhibitors of microgliaactivation.

Example 4 High Throughput Screen of a Test Compound Library

The SMDC/PHARMAKON (Microsource Discovery Systems, Gaylordville, Conn.)1600 Collection is a library of known drugs that have reacheddevelopment through clinical evaluation. Compounds from this library and307 additional test compounds (the “Test Compound Library”) were used inthe inhibition of microglial activation screens. The screens measuredthe ability of the various individual compounds within this TestCompound Library to inhibit fibrin-induced microglia activation.

Individual compounds from the Test Compound Library were added to afinal concentration of 10 μM to Fibrin-coated 384-well plates containingculture medium. After 1 hour of incubation of the plates in a 5% CO₂incubator at 37° C., primary rat microglia cells were plated at 6,000cells per well in a 384-well plate. Cells were fixed after 48 hours ofincubation in a 5% CO₂ incubator at 37° C. and labeled with CellMaskRed™ and the nuclear Hoechst dye. Images were acquired using the INCELLAnalyzer 2000 (GE Healthcare, United Kingdom) and analyzed with theINCELL Developer software, version 1.9. In the 384-well plate assayformat, two columns each were reserved for the negative control (6 μg/mlcoated Fibrin, 0% inhibition) and positive controls (uncoated wells,culture medium only, 100% inhibition), while 320 wells were used fortest compounds.

The assay performance for an initial screen of inhibition offibrin-induced microglia activation is shown in FIGS. 2 and 3. Z′ valuesmeasured were between 0.47 and 0.56 for the six assay plates (FIG. 2),and the amount of cell death within the normal range for both negativeand positive controls (FIG. 3).

The criteria for the selection of candidate therapeutic agents in thisexperiment were ≧50% inhibition of fibrin-induced microglial activationand less than 3% cell death to exclude toxic compounds. The results areshown in FIGS. 4 and 5. FIG. 4 is a plot showing the distribution ofpercent inhibition values for compounds and positive and negativecontrols. The screening of 1907 compounds resulted in the selection of128 candidate therapeutic agents in total which met the criteria for 50%or greater inhibition of fibrin-induced microglial activation and lessthan 3% cell death (a 7% hit rate) (FIG. 5). The 128 selected candidatetherapeutic agents were used to generate dose response curves forinhibition of microglial activation.

These test agents represent candidate therapeutic agents for theinhibition of microglia cell activation in vivo, as they demonstrateefficacy in the physiologically-predictive fibrin-induced activation ofmicroglia in the assay systems of the invention.

Example 5 Inhibition of LPS-Induced Microglia Activation

Primary rat microglia cells were plated into PDL-coated 384 well plates(Greiner, Germany) at 6,000 cells per well and incubated for an hour.After 1 hour, compounds from the Test Compound Library were added at aconcentration of 10 μM, 1 hour prior to the addition of LPS at aconcentration of 500 pg/ml. This concentration was chosen based on adose response curve for LPS-induced microglia activation as the optimaldose for maximum morphological activation. Cells were fixed after 48 hrsof incubation in a 5% CO₂ incubator at 37° C. and labeled with CellMask™Red Stain and nuclear Hoechst dye. Images were acquired using the INCellAnalyzer 2000 (GE Healthcare, United Kingdom) and analyzed with theINCELL-Developer software, version 1.9. The assay performance for thepilot screen of inhibition of LPS-induced microglial activation is shownin FIGS. 6 and 7. Z′ values were between 0.58 and 0.67 for the six assayplates (FIG. 6) and the amount of cell death within the normal range forboth negative and positive controls (FIG. 7).

The criteria for the selection of candidate therapeutic agents were ≧50%inhibition of LPS-induced microglial activation and less than 10% celldeath to exclude toxic compounds. The screening of 1907 test agents fromthe Test Compound Library resulted in 215 hits in total meeting the 50%inhibition criterion (an 11% hit rate). After removing test agents thatwere toxic, 146 compounds (a 8% hit rate) met the criteria for 50% orgreater inhibition of LPS-induced microglial activation and less than10% cell death (FIG. 8). These test agents represent candidatetherapeutic agents for the inhibition of microglia cell activation invivo, as they demonstrate efficacy in the physiologically predictiveLPS-microglia activation assay systems of the invention.

Example 6 Test Agents that Inhibit Both Fibrin-Induced and LPS-InducedMicroglia Activation Screens

FIG. 9 shows a distribution of test agents efficacious against both invitro fibrin-induced and LPS-induced microglia activation. Compoundsdistributing to the left top side of the graph were demonstrated to havegreater efficacy in inhibiting fibrin-induced microglia activation,whereas test agents on the right bottom were demonstrated to be moreefficacious in inhibiting LPS-induced activation. Several compounds inthe top right of the graph seem to equally inhibit both mechanisms ofactivation. Such test agents are particularly interesting as candidatetherapeutic agents for the inhibition of microglia cell activation invivo, as they demonstrate efficacy in two physiologically predictive invitro assay systems.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements and equivalents which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein.

What is claimed is:
 1. A cell-based assay for identification ofcandidate therapeutic agents that inhibit microglia activation in vivo,comprising: providing in vitro activated microglial cells; contactingsaid activated microglial cells with one or more test agents;identifying one or more test agents that exhibit inhibition of the invitro microglia activation; and determining toxicity of the identifiedtest agents that exhibit inhibition of the in vitro microglia activationby determination of cell death upon exposure of the microglial cells tothe test agents; wherein the test agents that exhibit both inhibition ofthe in vitro microglia activation and lack of toxicity are candidatetherapeutic agents for the inhibition of microglial activation in vivo.2. The assay of claim 1, further comprising the step of activatingmicroglial cells in vitro using an agent associated with microglialactivation.
 3. The assay of claims 1 and 2, wherein the microglial cellsare activated by in vitro exposure to an active fibrin composition. 4.The assay of claims 1 and 2, wherein the microglial cells are activatedby in vitro exposure to LPS.
 5. The assay of claim 1 through 4, whereinthe candidate therapeutic agents exhibit at least a 50% inhibition ofthe in vitro microglia activation.
 6. The assay of claims 1 through 5,wherein the toxicity is measured through identification of cell death ofthe activated microglial cells.
 7. The assay of claim 6, wherein thecell death is identified through shrinkage of the cell surface area ofthe microglia.
 8. The assay of claims 6 and 7, wherein the cell death isidentified through shrinkage of the cell surface area of the microgliato 150 μm² or less.
 9. The assay of claims 1 through 8, wherein the oneor more test agents comprise agents with demonstrated affinity totargets involved in microglia activation.
 10. The assay of claims 1through 9, wherein the one or more test agents comprise known drugs withclinical evaluation data.
 11. The assay of claims 1 through 10, whereinthe one or more test agents comprise known anti-inflammatory andmicroglia-macrophage response modulators.
 12. The assay of claims 1through 8, wherein the one or more test agents comprise agents with noknown activity or identified binding target.
 13. A high-throughputcell-based assay for identifying a candidate therapeutically activeagent for clinical inhibition of microglia activation, comprising thesteps of: providing a panel of in vitro activated microglial cells;contacting said panel of microglial cells with one or more individualtest agents; determining whether the test agents cause inhibition of theactivation of the microglial cells treated with the test agents; anddetermining the level of cell death of the microglial cells treated withthe test agents; wherein the one or more test agents that exhibitinhibition of the activation of the microglial cells and no increasedcell death are candidate therapeutically active agents for clinicalinhibition of microglia activation in vivo.
 14. The assay of claim 13,further comprising the step of activating microglial cells in vitrousing an agent associated with microglial activation.
 15. The assay ofclaim 14, wherein the microglial cells are activated by in vitroexposure to an active fibrin composition.
 16. The assay of claim 14,wherein the microglial cells are activated by in vitro exposure to LPS.17. The assay of claims 13 through 16, wherein the toxicity is measuredthrough identification of cell death of the activated microglial cells18. The assay of claim 17, wherein the cell death is identified throughshrinkage of the cell surface area of the microglia.
 19. The assay ofclaims 17 and 18, wherein the cell death is identified through shrinkageof the cell surface area of the microglia to 150 μm² or less.
 20. Theassay of claims 13 through 19, wherein the one or more test agentscomprise agents with demonstrated affinity to targets involved inmicroglia activation.
 21. The assay of claims 13 through 20, wherein theone or more test agents comprise known drugs with clinical evaluationdata.
 22. The assay of claims 13 through 21, wherein the one or moretest agents comprise known anti-inflammatory and microglia-macrophageresponse modulators.
 23. The assay of claims 13 through 19, wherein theone or more test agents comprise agents with no known activity oridentified binding target.